In gene therapy a carrier molecule called a vector must be used to deliver the therapeutic gene to the patient's target cells. Currently, the most common vector is a virus that has been genetically altered to contain therapeutic genetic sequences either to supplement expression of genes which are expressed in abnormally low levels or to inhibit expression of disease-causing genes. Target cells such as, for example, the patient's liver or lung cells are infected with the virus. The viral genome may then enter the nucleus of the cell and express the therapeutic sequence. If successful, gene therapy provides a way to fix a problem at its source. Adding a corrected copy of the gene may help the affected cells, tissues and organs work properly. Gene therapy differs from traditional drug-based approaches, which may treat the problem, but which do not repair the underlying genetic flaw.
One of the limitations of most gene therapies or vector-mediated RNA interference therapies delivered by viral or non-viral DNA vectors is that these therapies, once administered, are irreversible. Thus, the therapy could not be discontinued even if the patient were to have an unacceptable adverse reaction to the therapy. This inability for the switching gene therapy off may be detrimental or perhaps fatal to a patient.
It has been 15 years now that the Cre/lox system has been used as a way to regulate heterologous gene expression. The system begins with the cre gene, which encodes a site-specific DNA recombinase named Cre. Cre protein can recombine DNA when it locates specific sites in a DNA molecule. These sites are known as loxP sequences, which are 34 base pairs long and serve as substrates for the Cre-mediated recombination. When cells that have loxP sites in their genome also express Cre, the protein catalyzes a reciprocal recombination event between the loxP sites. Literally, the double stranded DNA is cut at both loxP sites by the Cre protein and then ligated back together. As a result, the DNA in between the loxP sites is excised and subsequently degraded.
This system has allowed researchers to create a variety of genetically modified animals and plants with the gene of their choice being externally regulated. For example, Sundaresan et al. have demonstrated that a PET reporter gene (PRG), the herpes simplex virus type 1 thymidine kinase (HSV1-tk), can be made to remain silent and can be activated by Cre-loxP-mediated recombination in cell culture and in living mice. Gene Ther., 11(7):609-18 (2004).
Recently, scientists have begun to recognize that the Cre-loxP system and other similar systems can be used for reversible gene therapy. For example, WO2005/112620 describes a gene therapy system comprising a Cre/loxP system. In their system, however, Cre is expressed in vivo. The expression of Cre in vivo often results in undesired expression of Cre due to leakage of the system. Such expression often results in unwanted genome rearrangements, and thus is unsafe for use in humans.
WO2005/039643, U.S. Patent Application Publication Nos. 20050130919, 20030022375, 20020022018, and 20040216178 also discloses either a Cre/loxP or an FLP/FRT system, but the application does not describe the use of exogenously applied Cre or use of a combination of Cre co-delivered with a cellular uptake enhancer.
U.S. Patent Application Publication No. 20030027335, discloses a Cre/loxP system with exogenously applied Cre but do not disclose that such system can be used to treat any human diseases.
Further, references fail to teach a system with multiple flanking sequences, thus failing to teach a system with added precision in modulating gene therapy as well as a safety shut-off of the system.
Thus, there continues to be a need in the art for novel compositions, kits, and methods of providing and regulating treatments delivered by gene therapy.